Approximately 20% of the populations of the industrialized world become hypersensitive (allergic) upon exposure to antigens from a variety of environmental sources. Those antigens that induce immediate and/or delayed types of hypersensitivity are known as allergens (Breiteneder, Hoffmann-Sommergruber et al. 1997). These include products of grasses, trees, weeds, animal dander, insects, food, drugs and chemicals. The antibodies involved in atopic allergy belong primarily to the immunoglobulin E isotype (IgE). IgE binds to basophils mast cells and dendritic cells via a specific high affinity receptor FcεRT. Upon exposure to an allergen, allergen-specific IgE antibodies on the cell surface become cross linked leading to the release of inflammatory mediators such as histamine and leukotrienes resulting in physiological manifestations of allergy (Akdis 2006).
Diagnostic tests for allergy involve the detection of IgE antibodies from patients with a specificity to proteins from an allergen source. However, a positive IgE test, i.e. IgE sensitisation, do not always lead to clinical manifestations of allergy and this discrepancy is one of the main reasons for trying to develop new and better diagnostic procedures. In typical tests, an aqueous extract from the allergen source, containing a mixture of proteins, is used in these tests. For most allergen sources, the allergenic proteins present in crude extract have only partly been identified and characterised. Diagnostic test procedures for detection of specific IgE antibodies in patients can either utilize an in vitro immunoassay using serum from the patient, or be a skin prick test (SPT), performed by topical application of the specific extract on the skin of the patient (Wainstein, Yee et al. 2007). In clinical practice, a doctor's diagnosis of allergy is usually based on both a positive test of IgE sensitisation for the relevant allergen source and a convincing clinical history of allergic reactions to this allergen. In recent years, many important allergenic proteins in the allergenic extracts have been identified and characterized. This has enabled the quantitation of specific IgE antibodies to each of these individual allergenic components, often referred to as component resolved diagnostics (CRD) (Hiller, Laffer et al. 2002) (Valenta, Lidholm et al. 1999) or molecular-based allergy (MA) diagnostics (Canonica, Ansotegui et al. 2013).
It is now widely recognised that Molecular-based allergy (MA) diagnostics has several distinct advantages as compared to conventional IgE analysis using allergen extracts (Canonica, Ansotegui et al. 2013). Analysis of all relevant allergen components from an allergen source has been shown to significantly increase the clinical utility of IgE testing as exemplified by wheat, peanut and hazelnut. (Nicolaou, Poorafshar et al. 2010; Codreanu, Collignon et al. 2011; Ebisawa, Moverare et al. 2012) (Masthoff, Mattsson et al. 2013). A necessary requirement if MA can be applied is that the majority of the individual allergen components from an allergen source has been identified and characterised.
One of the most important implications of MA diagnostics is to distinguish a genuine IgE sensitisation from sensitisation due to cross reactivity which may help the clinician to determine whether a single, a few closely related or several widely different allergen sources are responsible for the allergic symptoms. This can lead to an improved diagnosis of hypersensitivity of pollen (Stumvoll, Westritschnig et al. 2003), venoms (Müller, Schmid-Grendelmeier et al. 2012) and food allergy (Matsuo, Dahlstrom et al. 2008; Ebisawa, Shibata et al. 2012) In particular for peanut and hazelnut allergy, the use of allergen component IgE tests are better at predicting a clinical outcome of allergy than the use of a classical extract IgE test (Nicolaou, Poorafshar et al. 2010; Codreanu, Collignon et al. 2011; Masthoff, Mattsson et al. 2013). One reason for this is that some components may be low abundant and therefore only demonstrate IgE reactivity due to cross reactivity with homologous components from other species. Individuals having only IgE reactivity to such cross reactive components may therefore be less likely to have clinical symptoms to this allergen (Asarnoj, Moverare et al. 2010; Asarnoj, Nilsson et al. 2012). Despite their low clinical association, it is very important to identify all those low abundant cross reactive components in an allergen extract because in the clinical work up of a patient it is important that the sum of IgE reactivities to all components add up to that of the whole extract in order to rule out IgE reactivity to other minor or unknown components in the extract. The outcome of MA diagnostics can thus lead to improved selection of optimal immunotherapy treatment and better risk assessment of different food allergies.
Another use of allergen components is to use these to enhance the diagnostic sensitivity of an extract by spiking the extract with a component. This may be particularly important in miniaturized or non-laboratory immunoassay, such as an allergen microarray or a doctor's office test where the combination of less favourable assay conditions, lower capacity for antibody-binding allergen reagent and natural allergen extract of limited potency, may cause insufficient diagnostic sensitivity.
In conclusion, it is thus of great importance to identify and characterise all important allergenic proteins in each allergen source.
The treatment of allergy is most often reducing symptoms of allergy by e. g. anti-histamines but more long-term and curative treatment of allergy can be performed with specific immunotherapy. Application of the disease causing allergenic extract, most commonly either subcutaneously or sublingually, that causes a specific activation of a protective immune response to the allergenic proteins. Although the exact mechanisms are not fully known, such a specific activation of the immune system alleviates the symptoms of allergy upon subsequent environmental exposure of the same allergen (Akdis and Akdis 2007). A further development of regular immunotherapy has been to use one or several purified allergenic proteins instead of a crude natural extract. Such immunotherapy has been successfully performed for grass pollen allergic patients (Jutel, Jaeger et al. 2005) (Cromwell, Fiebig et al. 2006) (Saarne, Kaiser et al. 2005) and it has also been suggested for treating allergy against animal dander (Valenta, Lidholm et al. 1999; Gronlund, Saarne et al. 2009).
In recent years there has been increasing attention on allergen-specific IgG antibodies. These may modulate the effect of IgE antibodies, either directly by acting as blocking antibodies on the allergen or indirectly by acting via Fc receptors (Akdis and Akdis 2007; Uermosi, Beerli et al. 2010; Uermosi, Zabel et al. 2014).
Thus, by assessing both the specific IgE and the specific IgG response to an allergen may be more clinically relevant than measuring the IgE response alone (Custovic, Soderstrom et al. 2011; Caubet, Bencharitiwong et al. 2012; Du Toit, Roberts et al. 2015). It is well known that immunotherapy induces a specific IgG response which mainly consists of the IgG4 subclass. Since this antibody response is part of the mechanism for successful immunotherapy (Uermosi, Beerli et al. 2010; Uermosi, Zabel et al. 2014), the analysis-of allergen specific IgG antibodies may be a way to monitor the efficacy of the treatment.
In conclusion, the measurement of allergen-specific IgG levels may reflect natural or induced tolerance to the allergen through environmental exposure or immunotherapy treatment and may in combination with IgE levels increase the clinical relevance of a diagnostic test.
Horse dander is an increasingly common cause of respiratory allergy (Liccardi, D'Amato et al. 2011), with symptoms including rhinitis, conjunctivitis, bronchial inflammation and asthma. Occupational exposure to horse allergens is a significant risk factor for allergic sensitisation (Tutluoglu, Atis et al. 2002) but considerable concentrations of allergens can be detected also in other places such as schools (Kim, Elfman et al. 2005). IgE sensitisation to horse dander was in one study shown to be associated with a high risk of developing asthma (Ronmark, Perzanowski et al. 2003).
Extracts of horse hair and dander contain a complexity of allergenic proteins and four horse allergens have so far been identified: Equ c 1, Equ c 2, Equ c 3 and Equ c 4. The first two are both members of the lipocalin protein family and have been purified from their natural source (Dandeu, Rabillon et al. 1993; Goubran Botros, Rabillon et al. 1998) while only Equ c 1 has been expressed as a recombinant protein (Gregoire, Rosinski-Chupin et al. 1996). The amino acid sequence of Equ c 1 is 67% similar to that of the cat allergen Fel d 4 (Smith, Butler et al. 2004). Equ c 3, horse serum albumin, is a relatively conserved protein showing extensive cross-reactivity to other mammalian albumins (Goubran Botros, Gregoire et al. 1996). Equ c 4, was first purified (Goubran Botros, Rabillon et al. 1998; Goubran Botros, Poncet et al. 2001) and only later identified as horse sweat latherin (McDonald, Fleming et al. 2009). Recently, a novel horse allergen from the C-D subfamily of the secretoglobin protein family has been characterised (Equ c 15k, WO2011/133105).
Equ c 1 is claimed to be the most important one of the known horse allergens (Dandeu, Rabillon et al. 1993) and IgE antibody recognition of the recombinant protein was present in 76% of a population of horse allergic subjects studied (Saarelainen, Rytkonen-Nissinen et al. 2008). In another study using purified native allergens, only 33% of horse allergic patients were sensitized to Equ c 2 and 23% to Equ c 4 (Goubran Botros, Rabillon et al. 1998). The frequency of IgE binding to horse serum albumin has been addressed in several studies demonstrating reactivity in up to 40% of horse allergic subjects (Spitzauer et al. 1993; Cabañas et al. 2000). However, as sensitization to serum albumins is often accompanied by higher concentrations of IgE antibodies to other allergen components, its specific clinical relevance is uncertain (Spitzauer, Schweiger et al. 1993; Cabañas, López-Serrano et al. 2000). A recent study confirmed the relative prevalence of these horse allergen components and demonstrated a prevalence of 48% of the horse allergic patients having IgE reactivity to Equ c 15k. (WO2011/133105).